Bz 2 analyser

Excised hearts were fixed with 4% paraformaldehyde (PFA) in 0.1-M phosphate buffer containing 4% sucrose at 4 °C for >12 h. After a gradual dehydration with ethanol and xylene, hearts were embedded in paraffin and sectioned at 5 μm using a microtome (RM2155; Leica). The paraffin sections were deparaffinised by dipping into xylene and ethanol and then stained with Picrosirius Red for 1 h. After washing with 0.01-N HCl, sections were dehydrated with ethanol and xylene and mounted with Mount-Quick (Daido Sangyo). Sections were analysed using fluorescence microscopy (BioRevo BZ-9000; KEYENCE). The collagen volume fraction was calculated using a BZ-II analyser (KEYENCE). For post-MI day 3 samples, frozen sections of 6 µm thickness were prepared. The same method described above was used for Picrosirius Red staining.

Surgically resected tumor tissues were obtained from patients with ESCC from Osaka University Hospital (Osaka, Japan). Detailed methods are described in the online Supplementary Material.
Frozen sections were prepared from tumor tissues and stained for CD31 using a rat anti-mouse CD31 (BD Biosciences) followed by the Alexa-647-conjugated second antibody. Fluorescence images were captured using Biozero BZ-9000 (Keyence, Tokyo, Japan) in five random fields at 400× magnification. The fluorescence was quantitated by a standardized procedure using a BZ-II Analyser (Keyence).

Histological analysis were performed on snap-frozen tissue as previously described67 (link). In brief, sections were fixed with acetone for 10 min and nonspecific antigens were blocked in PBS containing 2% FCS for 15 min, followed by various stainings for 45 min.
Immunocytochemistry was performed on cells grown on coverslips50 (link). In brief, cells were fixed in 4% Formalin/PBS for 10 min, washed in PBS and then permeabilized in 1% Triton X–PBS for 10 min. After blocking with 10% FCS/PBS for 30–60 min, various stainings for 45 min were followed. Coverslips were mounted on microscope slides using mounting medium (S3023, Dako).
Images were acquired with a fluorescence microscope (KEYENCE BZ II analyser). Quantifications were performed using the Image J software (NIH).

Callus formation and bone maturation were assessed by means of histomorphometric analysis of Movat pentachrome and osteocalcin immunostained decalcified sections taken from the bone defect. For histological evaluation of bone maturation, bones were carefully defrosted and fixed in Zinc-Formal-Fixx, 10% over 20 h (Thermo Electron, Pittsburgh, USA) followed by decalcification for 14 days in 0.25 M Trizma base (Sigma-Aldrich, Taufkirchen, Germany) and 10% EDTA (Sigma-Aldrich), pH-value 7.4. Decalcified bones were paraffin embedded; longitudinal Sections (3 µm) were taken. Movat pentachrome staining of paraffin embedded histological slides was performed as published by Garvey et al. [31 (link)] using a staining kit according to the manufacturer’s instructions (Morphisto, Frankfurt, Germany). All slides were analysed using light microscopy. High resolution images depicting the whole defect zone in each case were created by automated stitching of multiple single frames covering the whole defect using the software BZII Analyser (Keyence, Neu-Isenburg, Germany). New bone formation and cartilaginous tissue area were then analysed in the defect site using the software ImageJ (https://imagej.nih.gov/ij/) and the relative tissue positive area of the entire defect zone was calculated.